Accurate diagnosis of active tuberculosis is required to improve treatment, reduce transmission of the disease and control the emergence of drug resistance. A rapid and reliable test would make a considerable contribution to the management of the TB epidemic, especially in HIV-burdened and resource-poor countries where access to diagnostic laboratories are limited. Surrogate marker antibody detection to mycobacterial lipid cell wall antigens gave promising results, in particular with cord factor. The specific advantage of using mycolic acids as lipid antigens in comparison to protein antigens is that mycolic acid is a CD1 restricted antigen with the ability to induce proliferation of CD4/CD8 double negative T-cells, which may explain the sustained antibody production in AIDS patients. Traditional end-point assays to detect anti-MA antibodies showed an unacceptable number of false positive and negative test results. Here a much improved biosensor method (the MARTI-assay, i.e. Mycolic acid Antibody Real-Time Inhibition assay) was developed to detect antibodies to mycolic acid in patient sera as surrogate markers of active tuberculosis. The test was assessed on an IAsys optical biosensor and gave an accuracy of 82%. The technology was transferred to an SPR (ESPRIT) biosensor to economise and simplify the assay. Mycolic acid containing liposomes were immobilized on the SPR gold surface pre-coated with octadecanethiol. The following parameters were optimized on the ESPRIT biosensor to enable reliable TB diagnosis: effect of degassed buffer, saponin blocking, first exposure to serum at low concentration and second exposure to antigen inhibited serum at high concentration. The IAsys biosensor system has a weakness in the double channel cuvette system, in which the channels often do not give matching results, while being ten times more expensive than the gold discs provided for the ESPRIT biosensor. The ESPRIT biosensor is provided with an adjustable laser setting to compensate for differences in the channel readings as well as an automated fluidic system that reduces variance from one sample to the next. First indications are that the test can also be used for prognosis of TB during treatment. It is hoped that the ESPRIT biosensor will improve the accuracy of the test to more than 90%. If the MARTI-assay technology could be made amenable for high throughput screening, it may provide the solution to the serodiagnosis of tuberculosis and monitoring of progress during TB treatment both in adult and children, thereby reducing the spread of TB within the communities. / Thesis (PhD)--University of Pretoria, 2009. / Biochemistry / unrestricted

Bacterial infections remain a significant threat to human health. Due to the emergence of widespread antibiotic resistance, development of novel antibiotics is required in order to ensure that effective treatment remains available. The aim of this study was to isolate and identify the fraction responsible for the antimicrobial activity in Tabernaemontana elegans (Stapf.) root extracts. The active fraction was characterised by thin layer chromatography (TLC) and gas chromatography – mass spectrometry (GC-MS). Antibacterial activity was determined using the broth micro-dilution assay and antimycobacterial activity using the BACTEC radiometric assay. Cytotoxicity of the crude extract and fractions was assessed against primary cell cultures; lymphocytes and fibroblasts; as well as a hepatocarcinoma (HepG2) and macrophage (THP-1) cell line using the Neutral Red uptake and MTT assays. The crude root extracts were found to contain a high concentration of alkaloids (1.2% w/w). GC-MS analysis identified the indole alkaloids, voacangine and dregamine, as major components. Antibacterial activity was limited to the Gram-positive bacteria and Mycobacterium species, with MIC values in the range of 64 – 256 ìg/ml. When combined with antibiotics, additive antibacterial effects were observed. Marked cytotoxicity to all cell lines tested was evident in the MTT and Neutral Red uptake assays, with IC50 values ranging between 1.11 – 9.81 ìg/ml. This study confirms the antibacterial activity of T. elegans and supports its potential for being investigated further for the development of a novel antibacterial compound. / Dissertation (MSc)--University of Pretoria, 2011. / Pharmacology / unrestricted

Background: Tobacco use is associated with exacerbation of tuberculosis (TB) and poor TB treatment outcomes. Integrating tobacco use cessation within TB treatment could improve healing among TB patients. The aim was to explore perceptions of health workers on where and how to integrate tobacco use cessation services into TB treatment programs in Uganda. Methods: Between March and April 2019, nine focus group discussions (FGDs) and eight key informant interviews were conducted among health workers attending to patients with tuberculosis on a routine basis in nine facilities from the central, eastern, northern and western parts of Uganda. These facilities were high volume health centres, general hospitals and referral hospitals. The FGD sessions and interviews were tape recorded, transcribed verbatim and analysed using content analysis and the Chronic Care Model as a framework. Results: Respondents highlighted that just like TB prevention starts in the community and TB treatment goes beyond health facility stay, integration of tobacco cessation should be started when people are still healthy and extended to those who have been healed as they go back to communities. There was need to coordinate with different organizations like peers, the media and TB treatment supporters. TB patients needed regular follow up and self-management support for both TB and tobacco cessation. Patients needed to be empowered to know their condition and their caretakers needed to be involved. Effective referral between primary health facilities and specialist facilities was needed. Clinical information systems should identify relevant people for proactive care and follow up. In order to achieve effective integration, the health system needed to be strengthened especially health worker training and provision of more space in some of the facilities. Conclusions: Tobacco cessation activities should be provided in a continuum starting in the community before the TB patients get to hospital, during the patients’ interface with hospital treatment and be given in the community after TB patients have been discharged. This requires collaboration between those who carry out health education in communities, the TB treatment supporters and the health workers who treat patients in health facilities.

Thesis (M. A. (Communication Studies)) -- University if Limpopo, 2021 / This study was prompted by the number of people who die daily from tuberculosis (TB) in the study area. The study investigated the perceptions of Mokopane residents, and their level of understanding and knowledge of the disease, tuberculosis (TB). The aim of the study was to profile people’s perceptions of health beliefs, the causes of the increase and spread of TB and its prevention and their knowledge about TB awareness campaigns in the Mokopane area. Data was collected by means of both qualitative and quantitative methods. Ten (10) medical doctors who operate private practices in the Mokopane area were interviewed. Data was collected from four hundred and one (401) participants from both Sandsloot and Tshamahansi villages outside Mokopane. The major findings of this study showed that firstly residents have a firm belief that traditional methods cure TB; secondly, they are largely ignorant of TB, its prevention, and consequences; and lastly that there is a lack of TB related education.

Mycobacterium tuberculosis (Mtb) is the causative agent of Tuberculosis (TB), a life-threatening disease primarily affecting the lungs that infects about one third of the world's population and causes 1.3 million deaths annually. It is estimated that TB has been infecting humans for around 70,000 years and has killed more people than any other infectious disease. The highly effective, persistent, and multifaceted virulence strategies that have allowed Mtb to continue to spread and thrive for so long are still poorly understood at the molecular level. This lack of knowledge contributes to ongoing challenges to curing TB. Although drugs capable of killing Mtb exist, even strains that are susceptible to these drugs remain so difficult to treat that stringent six- to nine-month courses of four-drug cocktails are required. Practical difficulties in administering full treatments and patient noncompliance have contributed to a rise in drug-resistant TB cases globally. To combat this increasing world health problem, new antibiotic treatments that kill Mtb and drug-resistant Mtb more effectively via new mechanisms of action are necessary. Discovering these antibiotics expediently requires that innovative Mtb-specific drug-screening assays are developed. An ideal and innovative TB drug screening method would target validated protein-protein interactions (PPI) essential to Mtb's pathogenesis and would be performed on whole Mtb cells under relevant in vivo-like conditions. This project focused on engineering several tools relevant to creating an ideal TB drug screen. A protein fragment complementation assay capable of studying PPI of the TB gyrase complex was created, and this assay was assessed for future HTS applications. To streamline the readout, this assay was re-engineered to include green fluorescent protein.

O estudo analisou a sustentabilidade da estratégia DOTS na visão de coordenadores de Programa de Controle Tuberculose (PCT) em sete municípios do interior do estado de São Paulo, prioritários para o controle da Tuberculose (TB). O quadro teórico está sustentado nas dimensões operacional, organizacional e política da gerência e sua articulação no contexto atual das políticas e serviços de saúde. A partir de uma abordagem quantitativa, de estudo epidemiológico descritivo por meio de levantamento de fontes secundárias, foram analisados os indicadores epidemiológicos do PCT: cura, abandono e óbito de casos novos com baciloscopia de escarro positiva, cobertura de Tratamento Supervisionado (DOT/TS) e de detecção de casos. Também optou-se pela abordagem qualitativa, análise de conteúdo-modalidade temática que utilizou como fonte a entrevista semi-estruturada com coordenadores de PCT. A Unidade Temática central foi a sustentabilidade da estratégia DOTS como um desafio para coordenadores de PCT, contendo dois sub-temas: A organização da estratégia DOTS diante da necessidade de captar/otimizar recursos e definir estratégias e A operacionalização das ações de controle da TB: raciocínio estratégico e negociação como ferramentas gerenciais. Os resultados quantitativos mostraram um coeficiente angular positivo para cura, mas a maioria não atingiu a meta de 85%; declínio no percentual de abandono, ainda aquém do esperado. A cobertura de DOT/TS apresentou aumento em todos os municípios, contudo, apenas um atingiu 95%. Nenhum município atingiu a meta de 70% de detecção de casos. Os temas que emergiram neste estudo apontaram nós críticos na captação e manutenção de incentivos financeiros; recursos humanos (RH) insuficientes e despreparados; rotatividade de pessoal; desconhecimento do destino da verba da TB e falta de autonomia para gerenciar os recursos; dificuldades de comunicação e integração com gestores; falta de priorização da doença na agenda política; priorização de agravos com repercussão política e necessidade de parcerias. Sobre a operacionalização do DOTS, o estudo apontou a cooperação de pessoas de fora do serviço como ferramenta gerencial estratégica, quando estimula a coresponsabilização da família e da comunidade como cuidadores ou multiplicadores. A avaliação, pautada em indicadores epidemiológicos, é utilizada prioritariamente para alcançar recursos, ao invés de subsidiar o planejamento. A gerência do PCT parece ser uma gerência burocrática, fundamentada no planejamento normativo. Acredita-se que a efetividade das ações gerenciais para sustentar a estratégia DOTS dependa de atores com conhecimento técnico, habilidades políticas e organizacionais, além de raciocínio estratégico para estimular e envolver todos os atores que lidam com a TB. Habilidades estas que, dificilmente são adquiridas na formação profissional. Conclui-se que os coordenadores trabalham em meio a pressões de cima e de baixo, ocupando dupla posição de transmissão, no plano hierárquico e das relações. Neste sentido, a sustentabilidade da estratégia DOTS representa desafios importantes para estes atores que utilizam poucos instrumentos gerenciais, têm pouca autonomia no processo decisório, lidam com recursos escassos e, muitas vezes, sem preparo para esta função, reforçando a necessidade de investimento na formação e capacitação contínua tanto para estes atores como para aqueles que lidam com a TB / This study analyzed the sustainability of the DOTS (Directly Observed Treatment Strategy). It shows the point of view of the TCP (Tubercolosis Control Program) coordinators of seven priority municipalities in the Sao Paulo State. The theorical framework is based on operational, organizational and political levels of the management and on its articulation in the current context of health policies and services. We carried out an epidemiological-descriptive study, using secondary sources, and through a quantitative approach. The following epidemiological TCP indicators were analyzed: cure, abandonment and death of new cases with positive sputum bacilloscopy, Supervised Treatment (DOTS/ST) and case detection coverage. We also chose to apply a qualitative approach, through a semi-structured interview with TCP Coordinators, and with the application of content analysis in the thematic modality. The main thematic unity is the sustainability of the DOTS strategy as a challenge to the TBP coordinators. It contains two sub-themes: The organization of the DOTS strategy faced with the need for resource captation/optimization and for clearly outlined strategies, and The operationalization of TB control actions: strategic thinking and the negociation as key management skills. Quantititive results show a positive angular coefficient for cure. Nonetheless, the majority did not achieve the 85% goal and the decline in the abandonment rate is still lower than expected. The ST coverage rose in all municipalities. However, only one municipality achieved coverage of 95%. None of the municipalities achieved the goal of 70% case detection rate. The themes analyzed in this study pointed to critical knots in the collection and maintenance of financial incentives; insuficient and unprepared Human Resources (HR); staff turnover; lack of knowledge concerning the destination of the TB funds, as well as lack of autonomy to manage resources; difficulties in the communication and in the integration with other managers; failure in making the disease a priority on the political agenda; priorization of aggravations with political repercussion and the need for partnerships. Concerning the DOTS operationalization, the study pointed to the cooperation of people outside the job as an important strategic management tool. It encourages family and community to take co-responsibility as caretakers and multipliers. The evaluation, which is based on epidemiological indicators, is especially used to achieve resources, instead of subsidizing the planning process. TCP management seems to be exceedingly bureaucratic and based on normative planning. We believe that the effectiveness of management actions to support the DOTS strategy needs characters with technical knowledge, political and organizational skills and strategic thinking. These skilss are important in order to encourage and get involved all those who deal with TB. These skills are hardly achieved in the professional training. We conclude that the coordinators work under pressure from the top and from the bottom of the hierarchy, and occupy a double transmission position: in the hierarchic level and in the relationship level. Thus, the sustainability of the DOTS strategy represents an important challenge for these characters, who use few management tools, have little authonomy in the decisionmaking process, have to work with scarce resources and are usually unprepared for the position they occupy. These findings highlight the need for more investments in continuing education and capacitation not only for coordinators but also for all those who have to deal with TB

O estudo analisou a sustentabilidade da estratégia DOTS na visão de coordenadores de Programa de Controle Tuberculose (PCT) em sete municípios do interior do estado de São Paulo, prioritários para o controle da Tuberculose (TB). O quadro teórico está sustentado nas dimensões operacional, organizacional e política da gerência e sua articulação no contexto atual das políticas e serviços de saúde. A partir de uma abordagem quantitativa, de estudo epidemiológico descritivo por meio de levantamento de fontes secundárias, foram analisados os indicadores epidemiológicos do PCT: cura, abandono e óbito de casos novos com baciloscopia de escarro positiva, cobertura de Tratamento Supervisionado (DOT/TS) e de detecção de casos. Também optou-se pela abordagem qualitativa, análise de conteúdo-modalidade temática que utilizou como fonte a entrevista semi-estruturada com coordenadores de PCT. A Unidade Temática central foi a sustentabilidade da estratégia DOTS como um desafio para coordenadores de PCT, contendo dois sub-temas: A organização da estratégia DOTS diante da necessidade de captar/otimizar recursos e definir estratégias e A operacionalização das ações de controle da TB: raciocínio estratégico e negociação como ferramentas gerenciais. Os resultados quantitativos mostraram um coeficiente angular positivo para cura, mas a maioria não atingiu a meta de 85%; declínio no percentual de abandono, ainda aquém do esperado. A cobertura de DOT/TS apresentou aumento em todos os municípios, contudo, apenas um atingiu 95%. Nenhum município atingiu a meta de 70% de detecção de casos. Os temas que emergiram neste estudo apontaram nós críticos na captação e manutenção de incentivos financeiros; recursos humanos (RH) insuficientes e despreparados; rotatividade de pessoal; desconhecimento do destino da verba da TB e falta de autonomia para gerenciar os recursos; dificuldades de comunicação e integração com gestores; falta de priorização da doença na agenda política; priorização de agravos com repercussão política e necessidade de parcerias. Sobre a operacionalização do DOTS, o estudo apontou a cooperação de pessoas de fora do serviço como ferramenta gerencial estratégica, quando estimula a coresponsabilização da família e da comunidade como cuidadores ou multiplicadores. A avaliação, pautada em indicadores epidemiológicos, é utilizada prioritariamente para alcançar recursos, ao invés de subsidiar o planejamento. A gerência do PCT parece ser uma gerência burocrática, fundamentada no planejamento normativo. Acredita-se que a efetividade das ações gerenciais para sustentar a estratégia DOTS dependa de atores com conhecimento técnico, habilidades políticas e organizacionais, além de raciocínio estratégico para estimular e envolver todos os atores que lidam com a TB. Habilidades estas que, dificilmente são adquiridas na formação profissional. Conclui-se que os coordenadores trabalham em meio a pressões de cima e de baixo, ocupando dupla posição de transmissão, no plano hierárquico e das relações. Neste sentido, a sustentabilidade da estratégia DOTS representa desafios importantes para estes atores que utilizam poucos instrumentos gerenciais, têm pouca autonomia no processo decisório, lidam com recursos escassos e, muitas vezes, sem preparo para esta função, reforçando a necessidade de investimento na formação e capacitação contínua tanto para estes atores como para aqueles que lidam com a TB / This study analyzed the sustainability of the DOTS (Directly Observed Treatment Strategy). It shows the point of view of the TCP (Tubercolosis Control Program) coordinators of seven priority municipalities in the Sao Paulo State. The theorical framework is based on operational, organizational and political levels of the management and on its articulation in the current context of health policies and services. We carried out an epidemiological-descriptive study, using secondary sources, and through a quantitative approach. The following epidemiological TCP indicators were analyzed: cure, abandonment and death of new cases with positive sputum bacilloscopy, Supervised Treatment (DOTS/ST) and case detection coverage. We also chose to apply a qualitative approach, through a semi-structured interview with TCP Coordinators, and with the application of content analysis in the thematic modality. The main thematic unity is the sustainability of the DOTS strategy as a challenge to the TBP coordinators. It contains two sub-themes: The organization of the DOTS strategy faced with the need for resource captation/optimization and for clearly outlined strategies, and The operationalization of TB control actions: strategic thinking and the negociation as key management skills. Quantititive results show a positive angular coefficient for cure. Nonetheless, the majority did not achieve the 85% goal and the decline in the abandonment rate is still lower than expected. The ST coverage rose in all municipalities. However, only one municipality achieved coverage of 95%. None of the municipalities achieved the goal of 70% case detection rate. The themes analyzed in this study pointed to critical knots in the collection and maintenance of financial incentives; insuficient and unprepared Human Resources (HR); staff turnover; lack of knowledge concerning the destination of the TB funds, as well as lack of autonomy to manage resources; difficulties in the communication and in the integration with other managers; failure in making the disease a priority on the political agenda; priorization of aggravations with political repercussion and the need for partnerships. Concerning the DOTS operationalization, the study pointed to the cooperation of people outside the job as an important strategic management tool. It encourages family and community to take co-responsibility as caretakers and multipliers. The evaluation, which is based on epidemiological indicators, is especially used to achieve resources, instead of subsidizing the planning process. TCP management seems to be exceedingly bureaucratic and based on normative planning. We believe that the effectiveness of management actions to support the DOTS strategy needs characters with technical knowledge, political and organizational skills and strategic thinking. These skilss are important in order to encourage and get involved all those who deal with TB. These skills are hardly achieved in the professional training. We conclude that the coordinators work under pressure from the top and from the bottom of the hierarchy, and occupy a double transmission position: in the hierarchic level and in the relationship level. Thus, the sustainability of the DOTS strategy represents an important challenge for these characters, who use few management tools, have little authonomy in the decisionmaking process, have to work with scarce resources and are usually unprepared for the position they occupy. These findings highlight the need for more investments in continuing education and capacitation not only for coordinators but also for all those who have to deal with TB

Philosophiae Doctor - PhD / The global impact of the converging dual epidemics of tuberculosis (TB) and human immunodeficiency virus (HIV) is one of the major public health challenges of our time, because in many countries, human immunodeficiency virus (HIV) and mycobacterium tuberculosis (TB) are among the leading causes of morbidity and mortality. It is found that infection with HIV increases the risk of reactivating latent TB infection, and HIV-infected individuals who acquire new TB infections have high rates of disease progression. Research has shown that these two diseases are enormous public health burden, and unfortunately, not much has been done in terms of modeling the dynamics of HIV-TB co-infection at a population level. In this thesis, we study these models and design and analyze robust numerical methods to solve them. To proceed in this direction, first we study the sub-models and then the full model. The first sub-model describes the transmission dynamics of HIV that accounts for behavior change. The impact of HIV educational campaigns is also studied. Further, we explore the effects of behavior change and different responses of individuals to educational campaigns in a situation where individuals may not react immediately to these campaigns. This is done by considering a distributed time delay in the HIV sub-model. This leads to Hopf bifurcations around the endemic equilibria of the model. These bifurcations correspond to the existence of periodic solutions that oscillate around the equilibria at given thresholds. Further, we show how the delay can result in more HIV infections causing more increase in the HIV prevalence. Part of this study is then extended to study a co-infection model of HIV-TB. A thorough bifurcation analysis is carried out for this model. Robust numerical methods are then designed and analyzed for these models. Comparative numerical results are also provided for each model. / South Africa

Thesis (MSc. (Microbiology)) -- University of Limpopo, 2019 / The emergence of drug resistance to the first line drugs complicates the treatment of tuberculosis (TB), especially in parts of sub-Saharan Africa where accessibility to quality health care is limited. The search for alternative medication has been the centre of research for years due to challenges posed by infectious organisms including drug resistance, lengthy treatment periods and lack of quality health care in developing countries. Stomatostemma monteiroae is used in traditional medicine to treat TB and related symptoms. The aim of this study was to isolate and characterise compounds with antimycobacterial activity from Stomatostemma monteiroae. The plant materials were collected from Ga-Madiga village in Limpopo province of South Africa. Different plant parts namely: leaves, twigs, roots, tuber and tuber-peels were separated, washed, dried and milled to a fine powder. Several solvents (n-hexane, dichloromethane, acetone and methanol) were used to extract the plant material using various extraction methods such as maceration, defatting, and extract enrichment procedure and phytochemical analysis was done using standard chemical tests and thin layer chromatography. The qualitative antioxidant activity was determined by the thin layer chromatography (TLC) based 2,2-diphenyl-1picrylhydrazyl (DPPH) free radical scavenging activity and quantitative antioxidant activity was determined using colorimetric DPPH free radical scavenging and ferric reducing power assay. Antimycobacterial activity of the extracts was assessed using bioautography and micro dilution method tested on Mycobacterium smegmatis (ATCC 1441), Mycobacterium tuberculosis (ATCC 25177) and M. tuberculosis H37Rv (ATCC 27294). The cytotoxic effects of the extracts were evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay on Vero monkey kidney cells. The compounds with antimycobacterial activity were isolated using bioassay-guided fractionation and purified using preparative thin layer chromatography and thereafter identified using NMR spectroscopy to elucidate the structure. Various phytochemical constituents were detected in different plant parts, with the leaves and twigs possessing more of the phytoconstituents analysed. The TLC profile of S. monteiroae indicated that more compounds are non-polar to intermediate in polarity. The antioxidant activity analysis on TLC plates indicated that all the plant parts have low antioxidant activity, this was also confirmed by xxii quantitative tests. The leaves of S. monteiroae had antimycobacterial activity when analysed using bioautography, while other plant parts had no active bands. The minimum inhibitory concentration values were much higher than the positive control rifampicin and the roots (0.31 mg/mL) followed by the leaves (0.83 mg/mL) had lower inhibitory concentrations when tested against M. smegmatis. The MIC values of extracts against TB causing strains varied greatly, the leaves and the roots had even higher MIC value. Toxicity analysis indicated that all plant parts were non-toxic towards Vero cells (LC50 > 0.02 mg/mL). Bioassay-guided fractionation enabled isolation of one antimycobacterial pure compound from the leaves extracts. The isolated compound was identified using NMR and was found to be a sitosterol derivative 8,9-dehydro-4-methyl-24-vinylobtusifoliol. This compound had a noteworthy activity against M. smegmatis. The present study validates the use of S. monteiroae in the treatment of TB related symptoms traditionally. Further studies are required to analyse the cytotoxic effects of the isolated compound and also testing the antimycobacterial activity of the isolated compound on TB causing pathogens. / National Research Foundation (NRF)

Mycolic acids are long chain lipids from the cell walls of Mycobacterium tuberculosis. The Nkuku phage display library was previously used to obtain monoclonal antibody binders to mycolic acids. In total 11 binders were obtained of which one was selected (MAC10) for further investigation by genetic engineering as presented in this dissertation. The antibodies of the Nkuku phage display library are in the format of single chain variable fragments (scFv). ScFv’s constitute only the epitope binding domains of an antibody consisting of the VH and VL domains fused into a single chain by a flexible linker protein. The selected anti-mycolic acid scFv is referred to as mycolic acid clone 10 (MAC10). Genes encoding the scFv’s of the Nkuku phage display library were cloned into the plasmid pHEN-1, a phage display vector. This vector is not commercially available or ideally suited for expression of scFv proteins. Therefore two vectors were investigated as possible targets for subcloning. The plasmids pGE20 and pAK400 were previously used for the expression of scFv antibody proteins. Subcloning into plasmid pAK400 proved to be the more efficient of the two investigated for subcloning. This subcloning yielded the recombinant plasmid pAKJS. Following the subcloning scFv protein expression was attempted using the plasmids pMAC10 (derived from pHEN-1) and pAKJS (derived from pAK400). Expression of MAC10 using plasmid pMAC10 in both Escherichia coli TG-1 and HB2151 was constitutive. This demonstrates that plasmid pHEN-1 is a non ideal vector as expression should not occur unless induced. Expression of MAC10 did not occur when pAKJS and Escherichia coli HB2151 were used. This was due to both the vector and expression host producing inhibitor protein for the Lac Z promoter controlling expression of the scFv. The MAC10 gene was subsequently randomized using the directed evolution method, error prone PCR. Sequence analysis of the five selected mutants indicated an average mutation rate of 8.6 mutations per 1000 base pairs. From the combined total of all five mutants, transversions made up the majority of substitutions. The majority of transversion mutations occurred at A-T base pairs. Transition substation mutations that made up the minority of total mutations occurred mostly at G-C base pairs. / Dissertation (MSc)--University of Pretoria, 2012. / Biochemistry / unrestricted